Reactive derivatives of sulforhodamine 101 with enhanced hydrolytic stability

ABSTRACT

The invention describes reactive dyes having an alkyl spacer attached via a sulfonamide bond to a sulforhodamine 101 fluorophore, and a variety of useful conjugates prepared therefrom. The increased length of the covalent linkage due to the alkyl spacer results in dye-conjugates having a number of surprisingly advantageous properties relative to previous sulforhodamine 101-labeled conjugates, including enhanced solubility and increased fluorescence. The reactive dyes of the present invention are more stable than the known compound sulforhodamine 101 sulfonyl chloride. Novel reactive dyes are described for selective modification of groups other than amines, including thiols and photoreactive derivatives.

This application is a division of application Ser. No. 08/485,033, filedJun. 7, 1995, now U.S. Pat. No. 5,798,276.

FIELD OF THE INVENTION

The invention relates to a family of fluorescent labeled conjugates ofsulforhodamine 101, and the chemically reactive fluorescent dyes thatare used to prepare those conjugates, including a wide range ofbiologically-derived or synthetic chemical materials.

BACKGROUND

Fluorescent dyes are known to be particularly suitable for biologicalapplications in which a highly sensitive detection reagent is desirable.Fluorescent dyes are used to impart both visible color and fluorescenceto other materials. Dyes that are able to preferentially bind to aspecific biological ingredient in a sample enable the researcher todetermine the presence or quantity of that specific ingredient. Inaddition, specific cellular structures can be monitored with respect totheir spatial and temporal distribution in diverse environments. Manyapplications utilize chemically reactive fluorescent dyes by chemicallyattaching the dye to reactive sites on a wide variety of materials suchas cells, tissues, proteins, antibodies, enzymes, drugs, hormones,lipids, nucleotides, nucleic acids, or natural or synthetic polymers tomake fluorescent conjugates.

Reactive derivatives of a wide variety of fluorescent dyes have beenpreviously described. This family of reactive dyes includes the sulfonylchloride derivative of sulforhodamine 101 (Titus et al. J. IMMUNOL.METH., 50, 193 (1982)), sold by Molecular Probes, Inc. under itsregistered trademark TEXAS RED. The sulfonyl chloride of sulforhodamine101 (hereafter referred to as SSC) is typically available as a mixtureof isomers, as shown below:

Conjugates of SSC with proteins and other biomolecules have achievedwide acceptance, particularly because their red fluorescence is readilydistinguishable from that of fluorescein (FIG. 2), with which they areoften combined in assays (Titus et al., supra).

Despite its many favorable characteristics, SSC possesses severaldisadvantages when used as a protein label. It is difficult to prepareprotein conjugates that have a high degree of SSC-labeling due to thestrong tendency of the resulting SSC-conjugates to precipitate fromsolution (Titus et al., supra; Waggoner et al., PRINCIPLES OF CLINICALFLOW CYTOMETRY, Chapter 7, pp 111-116). Furthermore, SSC is notappreciably soluble in water, necessitating prior dissolution of SSC inan organic solvent, which must then be added to the protein. Inaddition, the short spacer between SSC and the attachment site makesSSC-conjugates more likely to hinder the labeled probe in biologicalinteractions, such as with an enzyme, as when the SSC-labelednucleotides are used in conjunction with a nucleotide polymerase, orwith a ligand binding site, as in the case of a drug receptor. Finally,the fluorescence emission of SSC-labeled proteins tends to be quenched,relative to the fluorescence emission of the free dye.

In addition, the SSC dye itself possesses a sulfonyl chloride reactivesite, so it is intrinsically very susceptible to hydrolysis by eventrace amounts of water. This instability often results in labelingvariability, or total labeling failure. Sulfonyl chlorides also reactnon-selectively with groups in proteins other than amines, includingtyrosine, histidine and serine residues. Where these residues areessential for biological activity of the proteins, this reactivity isdetrimental to the use of the labeled protein. Furthermore, instabilityof these undesired adducts results in slow loss of the dye from theconjugates during storage.

The modification of SSC to form the dyes of the present invention allowsthe use of other less problematic reactive groups on the fluorophore,allowing conjugation with a wider range of substances. Derivatives ofsulforhodamine 101 that selectively react with functional groups otherthan amines (e.g. thiol-reactive dyes) were previously unknown. Anaminocaproic acid derivative of sulforhodamine 101 has recently beendescribed in the literature (Abuelyaman et al., BIOCONJUGATE CHEM. 5,400 (1994)), used as the hydroxybenzotriazole ester to prepare only aparticular sulforhodamine 101-labeled peptide phosphonate. The resultingintermediate activated ester is less chemically stable than analogousN-hydroxysuccinimidyl esters. Succinimidyl esters of aminoalkanoic acidsare known for other fluorophores, including tetramethylrhodamine(Haugland, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCHCHEMICALS, (1992)) and lissamine rhodamine (U.S. Pat. No. 5,393,514 toPitner et al., (1995)). These fluorophores possess a shorter wavelengththan those of the present invention, and they do not possess all of theadvantages of the reactive dyes of the invention, in particular, afluorescence emission that is well separated from that of fluorescein.

The novel dyes of the invention are derived from SSC, but they possessmany advantages that overcome the cited limitations of SSC, includinggreater water solubility, selective reactivity with a broader range offunctional groups, and considerably enhanced stability relative to SSC.The reactive dyes have sufficient chemical stability to permit theirpurification to a high degree of purity, including their separation intochemically pure single isomers, and the removal of trace amounts ofdisulfonyl chloride impurity, which is not practical for thehydrolytically unstable SSC. In addition, the conjugates that resultfrom the use of the new dyes retain or improve upon the beneficialproperties of labeling with sulforhodamine 101. Selected embodiments ofthe invention react with, and form conjugates with, substances havingreactive functional groups, including amines, thiols, alcohols andphenols. The conjugates of the present invention have fluorescenceemission wavelengths that are the same or longer in wavelength thanthose prepared from SSC, and thus have even less spectral overlap withthe emission of fluorescein (FIG. 2). Furthermore, the conjugatesprepared using the dyes of the present invention typically possess ahigher fluorescence quantum yield than SSC-conjugates, both as theresult of permitting a higher degree of substitution, and an unexpectedreduction of quenching of the dyes by their conjugates (FIG. 7). Theprotein-conjugates of the present invention are unexpectedly much moresoluble than those prepared using SSC, and are less prone toprecipitation. Finally, the dyes of the present invention labeloligonucleotides more efficiently than SSC, and the resultingoligonucleotide conjugates are more readily purified.

DESCRIPTION OF DRAWINGS

FIG. 1: The normalized fluorescence emission spectra of selectedconjugates of streptavidin. A) rhodamine-x isothiocyanate, B)SSC-streptavidin and C) Compound 2-streptavidin conjugate (Example 19).

FIG. 2: The normalized fluorescence emission spectra of selectedconjugates of goat IgG antibody. A) fluorescein, B) SSC, and C) Compound2 (Example 19).

FIG. 3: The normalized absorption spectra of selected conjugates ofstreptavidin in PBS at pH 7.2: A) rhodamine-X isothiocyanate (XRITC), B)SSC, C) Compound 2.

FIG. 4: The normalized absorption spectra of goat anti-mouse antibodyconjugates of A) SSC, and B) Compound 2 (Example 19).

FIG. 5: A comparison of total fluorescence as a function of the degreeof fluorophore substitution for streptavidin conjugates of XRITC, SSCand Compound 2 (Example 23).

FIG. 6: A comparison of the fluorescence emission of conjugates of2′-deoxyuridine-5′-triphosphate (Example 31). A) SSC-dUTP; B) Compound2-dUTP.

SUMMARY OF THE INVENTION AND DESCRIPTION OF PREFERRED EMBODIMENTS

Reactive dyes of the invention have an alkyl spacer attached by asulfonamide bond to a sulforhodamine 101 fluorophore. The increasedlength of the covalent linkage due to the alkyl spacer results indye-conjugates having a number of surprisingly advantageous propertiesrelative to previous sulforhodamine 101-labeled conjugates, includingenhanced water solubility and increased fluorescence. The reactive dyesof the present invention are more stable than the known compoundsulforhodamine 101 sulfonyl chloride (“SSC”). Novel reactive dyes aredescribed for selective modification of amines, as well as other groups,including thiols and photoreactive derivatives.

Reactive Dyes

The reactive dyes of the present invention are all derivatives preparedfrom SSC. As with SSC, the reactive dyes of the invention are typicallyavailable as a mixture of the two mono-sulfonyl isomers ofsulforhodamine 101, typically containing some disulfonyl derivative.While the parent material, SSC, is insufficiently stable to permit apractical separation of the isomers and contaminants, the reactive dyesof the present invention are typically stable enough for the isomers tobe purified by conventional means, including column chromatography andhigh performance liquid chromatography (HPLC). While the two isomers aregenerally equivalent for many of the purposes of the present invention,purification of discrete isomers is sometimes required for the mostcritical assays. It is understood that while the “para” sulfonamideisomer is typically shown and referred to, unless specifically statedotherwise, each isomer as well as mixtures of both isomers areencompassed by the present invention.

The reactive dyes of the present invention have the general formula

or the general formula

For all dyes, R² is H, C₁-C₆ alkyl, or C₁-C₆ acyl (—(C═O)—R′, where R′is H or C₁-C₅ alkyl); preferably R² is H, methyl or acetyl. For alldyes, n=1-8 and R is a reactive group that spontaneously reacts with anappropriate functional group to yield a covalent linkage.

For one class of reactive dyes, n=2to 8, and the reactive group R is ahaloacetamide (—NH—(C═O)—CH₂—X) or a halomethylbenzamide(—NH—(C═O)—C₆H₄—CH₂—X), where X is Cl, Br or I. Alternatively, R is amaleimide,

a maleimidyl benzamide

a maleimidyl alkylamido,

an azidobenzamido,

an azidoperfluorobenzamido,

a (3,5-dichloro-2,4,6-triazin-1-yl)amino,

an isocyanato (—N═C═O), or an isothiocyanato (—N═C═S).

In another embodiment of the invention, n is 1 to 7, and the reactivegroup R is a carboxylic acid (—COOH), or a derivative of a carboxylicacid. An appropriate derivative of a carboxylic acid includes an alkalior alkaline earth metal salt of a carboxylic acid. Alternatively, R is areactive derivative of a carboxylic acid (—(C═O)—R_(x)), where thereactive group R_(x) is one that activates the carbonyl group of—(C═O)—R_(x) toward nucleophilic displacement. In particular, R_(x) isany group that activates the carbonyl towards nucleophilic displacementwithout being incorporated into the final displacement product.

Typically, R_(x) is a good leaving group, selected so as to make R anactivated ester of a carboxylic acid: R is optionally a symmetricanhydride that links two sulforhodamine 101 fluorophores, or a simplemixed anhydride of a sulforhodamine 101 fluorophore and a C₂-C₈chloroformate, a C₂-C₈ carboxylic acid or perfluorinated carboxylicacid, a C₁-C₈ sulfonic or fluorinated sulfonic acid. Alternatively, R isan acyl azide. R is alternatively a carboxylic acid activated by acarbodiimide having 2-14 carbons. Finally, R is an ester of a phenol ora naphthol that is further substituted by at least one strong electronwithdrawing group. Selected electron withdrawing groups, present in anycombination, include but are not limited to nitro, sulfo, carboxy,alkali or alkaline earth metal salt of sulfo or carboxy, cyano, fluoroor chloro, or trifluoromethyl. Particularly suitable substituted arylesters include nitrophenyl, sulfophenyl, pentafluorophenyl andpentachlorophenyl esters. Additional R groups include acyl nitrites oracyl hydrazides.

While a variety of activated carboxylic acids and activated esters aresuitable for preparing the conjugates of the invention, some areextremely reactive. While a very reactive dye is advantageous for facilepreparation of conjugates, they must often be prepared and utilized insitu, as they are not stable enough to be isolated as pure compounds.This class of dyes includes those for which the R_(x) group is chlorideor fluoride, yielding an acid halide. In particular, the class ofreactive dyes wherein R is a 1-hydroxybenzotriazole ester are generallytoo reactive to isolate, although this method of protein conjugation isknown in the art. Activated carboxylic acids wherein R is a succinimidylester or a sulfosuccinimidyl ester, or the alkali or alkaline earthmetal salt of these esters, possess greatly enhanced stability, relativeto 1-hydroxybenzotriazole esters, and may be isolated, stored andseparated into pure isomers.

Preferred R groups are those that react spontaneously with thefunctional groups on a biomolecule or other substance, without requiringthe presence of a third reagent (such as a catalyst) or unusually harshconditions, such as very high or low pH, extremes in temperature,organic solvents or other factors.

Synthesis

Reactive dyes that are amine-reactive are generally prepared by thereaction of SSC with an amino acid or its ester-protected derivative(Example 1). The resulting isomers may be separated by chromatographicmeans at this step or following removal of any protecting groups (e.g.for use as a derivatization reagent in ultra-high resolution separationtechniques such as gel or capillary electrophoresis) however, separationis not required or preferred. The dye is converted to an amine-reactivederivative by methods well recognized in the art, such as by conversionto a succinimidyl ester (Example 2), a sulfosuccinimidyl ester (Example3) or an acyl azide (Example 12). Alternatively, the dye is activated insitu prior to coupling to an amine, for instance by reaction with awater-soluble carbodiimide (Example 3).

The synthesis of haloacetamides, halomethylbenzamides, maleimides,azidobenzamides and 3,5-dichloro-2,4,6-triazines typically utilizes asulforhodamine 101 sulfonyl cadaverine conjugate, the structure of whichis shown below, which is commercially available (TEXAS REDsulfonylcadaverine, Molecular Probes, Eugene Oreg.). Alternatively, anyof the C₂-C₈ homologs of sulforhodamine 101 sulfonyl cadaverine may beused as a starting material (Examples 7-10).

The isocyanate and isothiocyanate derivatives of sulforhodamine 101 areprepared by reaction of sulforhodamine 101 cadaverine (or one of itshomologs) with phosgene or thiophosgene, or one of their syntheticequivalents and alternatives, as well documented in the art. Preferably,the isocyanate derivative is synthesized by the Curtius rearrangement ofthe corresponding acyl azide (Example 12).

Dye-Conjugates

The reactive dyes of the invention are used to label organic substancesto form dye-conjugates by the intermolecular reaction of the reactivegroup on the reactive dye with an appropriate functional group on theorganic substance to be conjugated. Appropriate organic substances forconjugation can either be isolated from natural products, preparedsynthetically, or isolated from a natural product and then syntheticallymodified (semi-synthetic). The reactive dyes can label a wide variety oforganic substances, provided that the organic substance contains afunctional group that possesses suitable reactivity with any one of thereactive groups, R, that are described above. Useable functional groupson the organic substance include, but are not limited to, amines,thiols, alcohols, phenols, aldehydes, ketones, phosphates, imidazoles,hydrazines, hydroxylamines, disubstituted amines, halides, epoxides,sulfonate esters, purines, pyrimidines, or carboxylic acids. Amines,thiols, and alcohols are the preferred functional groups forconjugation, as they are both more reactive and more commonly availablefor the modification of biomolecules. However, a wide variety of otherfunctional groups react under conditions well understood by one skilledin the art (as listed in Table 1). The latter include hydrazinederivatives, hydroxylamine derivatives, thioethers, di- andtrisubstituted amines, and carboxylic acids (to form esters). Thefunctional group on the organic substance may be attached directly, orattached via any useful spacer or linker. A dye-conjugate is preparedfrom either a readily-available organic substance, or from an initiallynon-reactive organic substance that has been derivatized by anappropriate functional group (as above).

TABLE 1 Examples of some routes to useful conjugations FUNCTIONAL GROUPYIELDING: REACTIVE GROUP R (attached to (type of (attached to reactivedye) organic substance) conjugation) haloacetamides thiols thioethersmaleimides thiols thioethers alkyl halides thiols thioethers alkylsulfonates thiols thioethers alkyl halides alcohols/phenols ethers alkylsulfonates alcohols/phenols ethers carboxylic acids amines/anilinescarboxamides anhydrides amines/anilines carboxamides activated esters*amines/anilines carboxamides chlorotriazines amines/anilinesaminotriazines isocyanates amines/anilines ureas isothiocyanatesamines/anilines thioureas sulfonyl halides amines/anilines sulfonamidesalkyl halides amines/anilines alkyl amines sulfonate estersamines/anilines alkyl amines *as described previously

Dyes that are selected to conjugate with substances or materials havingfree amine groups are preferably those dyes of the invention for which Ris a succinimidyl or sulfosuccinimidyl ester. Preferably, amine-reactiveembodiments have n=4 or 5. Amine-reactive dyes are of particularrelevance as they are commonly used to label proteins and polypeptides,which possess free amine groups. Amine-reactive dyes are additionallyused to label materials that have been substituted with free aminegroups, such as amino-dextrans, or amine containing nucleotides,oligonucleotides or nucleic acids.

Dyes that are selected to conjugate with materials having free thiolgroups are preferably those dyes of the invention for which R is ahaloacetamide, halomethylbenzamide, or a maleimido group. Morepreferably, R is an iodoacetamide, maleimido, maleimidylacetamide or ahalomethylbenzamide. Preferably, thiol-reactive embodiments have n=5 or6.

Preferred alcohol- and phenol-reactive dyes are those dyes of theinvention for which R is an isocyanate, 3,5-dichloro-2,4,6-triazine,acyl nitrile or is a phosphoramidite. Preferably, alcohol-reactiveembodiments have n=5 or 6.

Preferred photoreactive dyes have n=5 or 6, and R is anazidoperfluorobenzamido group.

In one embodiment of the invention, the conjugated substance is an aminoacid, peptide, or protein. By amino acid is meant any of the naturalamino acids, as well as synthetic variations commonly known and utilizedin the art. Common synthetic variations include amino acids that areprotected on their amino, carboxylic acid, hydroxy or other functionalgroup. Both peptides and proteins fall under the general category ofpeptides. While the specific demarcation line between peptides andproteins is not exact, it is typically recognized in the art thatpeptides have molecular weights of less than about 5,000 to 10,000daltons, and proteins have molecular weights greater than about 5,000 to10,000 daltons. Although peptides include molecules as small asdipeptides, the preferred peptides of the invention contain at leastfive amino acids, more preferably 5 to 36 amino acids. Preferredpeptides of the invention include neuropeptides, chemotactic peptides,gastrointestinal peptides, snake toxins, protease substrates,endothelin, protein kinase substrates and others. Proteins typicallypossess at least secondary structure, and most often tertiary andquaternary structure. The protein conjugates of the present inventiontend to be more soluble, and display less fluorescence quenching thanthe previously known SSC-protein conjugates.

The protein conjugates of the present invention encompass a variety ofproteins, including but not limited to enzymes, antibodies, lectins,glycoproteins, lipoproteins, avidin, streptavidin, protein A, protein Gand phycobiliproteins. By enzyme is meant any of a group of catalyticproteins that are produced by living cells and that mediate and promotethe chemical processes of life without themselves being altered ordestroyed. Examples of appropriate enzymes suitable for conjugationinclude, but are not limited to, peroxidases, proteases, phosphatases,and blycosidases, such as b-D-galactosidases, and b-D-glucuronidases.Antibodies, as used herein, are any of various proteins synthesized byanimals in response to the presence of a foreign substance, for example,Immunoglobulin G (IgG) and its fragments. Lectins, as used herein, areany of various proteins that selectively bind carbohydrates, such ascell surface carbohydrates, which can be used to identify cell type.Appropriate lectins are typically isolated from plants, preferablylegumes, or from bacteria, fish or invertebrates. A preferred lectin iswheat germ agglutinin. Glycoproteins, as used herein, are any of a classof conjugated proteins containing both carbohydrate and protein units.Phycobiliproteins are any of several proteins isolated from algae,including but not limited to b-phycoerythrin, R-phycoerythrin,C-phycocyanine or allophycocyanin.

In another embodiment of the invention, the conjugated substance is asingle base, single nucleoside, single nucleotide or a nucleic acidpolymer. By nucleotide is meant the basic structural unit of a nucleicacid, comprising an ester of a nucleoside and one or more phosphoricacid or polyphosphoric acid groups, optionally containing an additionallinker or spacer for attachment of a fluorophore or other ligand, suchas an alkynyl linkage (U.S. Pat. No. 5,047,519 to Hobbs, Jr. et al.,(1991), incorporated by reference) or other linkage (U.S. Pat. No.4,711,958 to Ward et al., (1987); U.S. Pat. No. 5,175,269 toStavrianopoulos, (1992); U.S. Pat. No. 5,241,060 to Engelhardt et al.,(1993); U.S. Pat. No. 5,328,824 to Ward et al., (1994); all of which arehereby incorporated by reference). The conjugated nucleotide istypically a ribonucleotide, deoxyribonucleotide or adideoxyribonucleotide. Preferably, the conjugated nucleotide is mono-,di- or triphosphate ester of adenosine, guanosine, uridine or cytidine.More preferably, the conjugated nucleotide is uridine triphosphate ordeoxyuridine triphosphate.

Nucleic acid polymers are typically large, chainlike moleculescontaining phosphoric acids, sugars, and purine and pyrimidine bases.Polymers that are oligonucleotides are typically composed of fewer than50 nucleotides, more typically composed of fewer than 25 nucleotides.Oligonucleotides are optionally deoxyribonucleic acid polymers (DNA) orribonucleic acid polymers (RNA), or a hybrid thereof. Suitableoligonucleotides are optionally antisense oligonucleotides, or strandsof DNA having a sequence identical to messenger RNA. DNA polymers areoptionally single-stranded (ss), double-stranded (ds), triple-strandedor quadruple-stranded DNA. RNA is optionally single-stranded ordouble-stranded nucleic acid polymers. The nucleic acid polymer may be anatural polymer (biological in origin) or a synthetic polymer (modifiedor prepared artificially). The nucleic acid polymer optionallyincorporates an unusual linker such as morpholine derivatized phosphates(AntiVirals, Inc., Corvallis Oreg.), or peptide nucleic acids such asN-(2-aminoethyl)glycine units (Wittung, et al., NATURE 368, 561 (1994)).In one embodiment of the invention, the dye is attached to thenucleotide, oligonucleotide or nucleic acid polymer via one or morepurine or pyrimidine bases through an amide, ester, ether or thioetherbond. In another embodiment of the invention, the dye is attached to thephosphate or carbohydrate by a bond that is an ester, thioester, amide,ether or thioether. In one embodiment of the invention, where theconjugated substance is a nucleotide, the reactive group R on thereactive dye is a carboxylic acid, a derivative of a carboxylic acid, oran activated ester of a carboxylic acid. Preferably, the reactive groupR is a succinimidyl ester or a sulfosuccinimidyl ester.

In another embodiment of the invention, the conjugated substance is acarbohydrate. By carbohydrate is meant any of the group of organiccompounds composed of carbon, hydrogen and oxygen, including sugars,starches and celluloses. In particular, carbohydrates includespolysaccharides such as dextran, FICOL, heparin, glycogen, amylopectin,mannan, inulin, starch, agarose and cellulose. All of thesepolysaccharides are readily available at low cost, high purity, lowbackground absorbance and fluorescence and have relatively uniformphysical properties. Preferably a carbohydrate conjugate is a dextran orficol conjugate, more preferably a dextran conjugate.

In another embodiment of the invention, the conjugated substance is alipid. By lipid is meant one of a class of compounds that containslong-chain aliphatic hydrocarbons and their derivatives, such as fattyacids, alcohols, amines, amino alcohols, and aldehydes. The class oflipids include glycolipids, phospholipids and sphingolipids. Glycolipidsare lipids that contain carbohydrate units. Phospholipids are lipidscontaining esters of phosphoric acid containing one or two molecules offatty acid, an alcohol, and generally a nitrogenous base. Sphingolipidsare lipids, such as sphingomyelin, that yield sphingosine or one of itsderivatives as a product of hydrolysis. Alternatively, the conjugatedsubstance is a lipid vesicle.

One class of conjugates of the present invention includes conjugates ofbiologically active molecules. Biologically active molecules include,but are not limited to, cytokines such as lymphokines, hormones,steroids, toxins, or drugs. Alternatively, conjugates of the presentinvention are conjugates of members of a specific binding pair, such asan antigen or a hapten. In another embodiment, the instant conjugatesare conjugates of metabolites, or environmental pollutants.

Another class of conjugates included in the present invention includesconjugates of colorimetric or fluorescent dyes, including but notlimited to fluoresceins, rhodamines, resorufins, dipyrrometheneborondifluorides, coumarins, carbocyanines, fluorescent proteins or otherfluorophore groups. As an example, the conjugate of phycoerythrin usingCompound 2 of the present invention displays an essentially completeenergy transfer to the sulforhodamine 101 emission band when thephycoerythrin is illuminated (Example 20). Bifluorophoric dyescomprising dyes of the present invention and other colorimetric orfluorescent dyes will also undergo excited state energy transfer if thetwo fluorophores possess suitably overlapping spectra and are in closeproximity, i.e. where the distance between the fluorophore is about 50Å.

Alternatively, the conjugates of the present invention are conjugates ofcellular systems, cellular fragments, or subcellular particles. Examplesof this type of conjugated material include virus particles, bacterialparticles, virus components, biological cells, or cellular components.Examples of cellular components that can be labeled, or whoseconstituent molecules can be labeled, include lysosomes, endosomes,cytoplasm, nuclei, mitochondria, Golgi apparatus and vacuoles.

Finally, the conjugates of the present invention are optionallydye-conjugates of polymers, polymeric particles, polymeric membranes,conducting and non-conducting metals and non-metals, and glass andplastic surfaces and particles.

Conjugates of most low molecular weight drugs, peptides, toxins,nucleotides, phospholipids and other organic molecules are prepared byorganic synthesis methods using the reactive dyes of the invention, bymeans well recognized in the art (Haugland, MOLECULAR PROBES HANDBOOK,supra, Sets 1-7, (1992)). Preferably, conjugation to form a covalentbond consists of simply mixing the reactive dyes of the presentinvention in a suitable solvent in which both the reactive dye and thesubstance to be conjugated are soluble. The reaction preferably proceedsspontaneously without added reagents at room temperature or below. Forthose reactive dyes that are photoactivated, conjugation requiresillumination of the reaction mixture to activate the reactive dye.Chemical modification of water insoluble substances, so that a desireddye-conjugate may be prepared, is preferably performed in an aproticsolvent such as dimethylformamide, dimethylsulfoxide, acetone, ethylacetate, toluene, or chloroform. Similar modification of water-solublematerials is readily accomplished through the use of the instantreactive dyes to make them more readily soluble in organic solvents.Particularly useful for labeling substances in aqueous solutions are thesulfosuccinimidyl esters of the present invention (Example 3).

Conjugates of polymers, including biopolymers and other higher molecularweight polymers are typically prepared by means well recognized in theart (for example, Brinkley et al., BIOCONJUGATE CHEM., 3, 2 (1992)). Inthese cases, a single type of reactive site may be available, as istypical for polysaccharides) or multiple types of reactive sites (e.g.amines, thiols, alcohols, phenols) may be available, as is typical forproteins. Selectivity of labeling is best obtained by choice on anappropriate reactive dye. For example, modification of thiols with athiol-selective reagent such as a haloacetamide or maleimide, ormodification of amines with an amine-reactive reagent such as anactivated ester, acyl azide, isothiocyanate or3,5-dichloro-2,4,6-triazine. Partial selectivity can also be obtained bycareful control of the reaction conditions.

Where dye-conjugates are prepared using a photoreactive dye of theinvention, such as an azidoacyl derivative, the conjugation requiresillumination of the dye by light having a suitable wavelength, typically<400 nm.

When modifying polymers with the dyes, an excess of dye is typicallyused, relative to the expected degree of dye substitution. Any residual,unreacted dye or a dye hydrolysis product, is typically removed bydialysis, chromatography or precipitation (Example 19). Presence ofresidual, unconjugated dye can be detected by thin layer chromatographyusing a solvent that elutes the dye but not its polymer conjugate. Inall cases it is usually preferred that the reagents be kept asconcentrated as practical so as to obtain adequate rates of conjugation.When the substance to be conjugated is a protein, the preferred proteinconcentration is 1 to 10 mg/mL.

For soluble dye-conjugates that have multiple attachment sites, thedegree of substitution of the polymer is typically determined by firstdissolving the dye-free unlabeled polymer in a suitable solvent, andmeasuring its long wavelength absorption. A determination of thelong-wavelength absorption of the labeled dye-conjugate can then be usedto determine the approximate degree of substitution of the conjugate,given an approximate value for the extinction coefficient of thesulforhodamine 101 fluorophore. The value that is typically used for theextinction coefficient for the dyes of the present invention for thiscalculation is 80,000 cm⁻¹M⁻¹.

In one aspect of the invention, the conjugate of the invention isassociated with an additional substance, that binds to either thesulforhodamine 101 fluorophore or the conjugated substance throughnoncovalent interaction. In a specific embodiment, the additionalsubstance is an antibody, a lectin, a receptor, an oligonucleotide, anucleic acid, or a polymer. The additional substance is optionally usedto probe for the location of the dye-conjugate, for example, as a meansof enhancing the signal of the dye-conjugate.

In another embodiment of the invention, one of the reactive dyes of theinvention is provided with instructions for conjugating the dye to anysubstance possessing an appropriate functional group, and optionally forrecovering or purifying the materials labeled thereby. This combinationof reactive dye and instructions therefore comprise a kit for labeling aspecific substance. In selected embodiments of the invention, the kitthereby formed would possess utility for labeling proteins,oligonucleotides or carbohydrates. The dyes of the present invention arewell-suited for the preparation of such a kit, as they possess greatlyenhanced stability with respect to SSC, and can therefore more readilybe shipped and stored without loss of reactivity.

Applications of the Dye-Conjugates

Typically, the dye-conjugate is a labeled member of a specific bindingpair, and is used as a fluorescent probe for the complementary member ofthat specific binding pair. A specific binding pair member can be aligand or a receptor. As used in this document, the term ligand meansany organic compound for which a receptor naturally exists or can beprepared. A receptor is any compound or composition capable ofrecognizing a spatial or polar organization of a molecule, e.g. epitopicor determinant site. Ligands for which naturally occurring receptorsexist include natural and synthetic proteins, including avidin andstreptavidin, antibodies, enzymes, and hormones; nucleotides and naturalor synthetic oligonucleotides, including primers for RNA and single- anddouble-stranded DNA; lipids; polysaccharides and carbohydrates; and avariety of drugs, including therapeutic drugs and drugs of abuse andpesticides. Ligands and receptors are complementary members of aspecific binding pair, each specific binding pair member having an areaon the surface or in a cavity which specifically binds to and iscomplementary with a particular spatial and polar organization of theother. Representative specific binding pairs are shown in Table 2.

TABLE 2 Representative Specific Binding Pairs antigen antibody biotinavidin (or streptavidin) IgG* protein A or protein G drug receptor drugtoxin receptor toxin carbohydrate lectin peptide receptor peptideprotein receptor protein carbohydrate receptor carbohydrate DNA (RNA)aDNA (aRNA)^(†) *IgG is an immunoglobulin ^(†)aDNA and aRNA are theantisense (complementary) strands used for hybridization

In one aspect of the invention, the specific binding pair member is anantibody or antibody fragment, avidin or streptavidin. In thisembodiment of the invention, the complementary binding pair member istypically a hapten, an antigen or a biotin. Where the complementarybinding pair member is a hapten, the hapten typically has a molecularweight less than 1,000. In another aspect of the invention, the specificbinding pair member is an oligonucleotide or nucleic acid polymer.Optionally, the complementary binding pair member is present in a cell,bacteria, virus or yeast cell. Alternatively, the complementary memberis immobilized on a solid or semi-solid surface, such as a polymer,polymeric membrane or polymeric particle (such as a latex bead).

Preferably, the fluorescent conjugate of a specific binding pair memberis useful for detecting and optionally quantifying the presence of thecomplementary specific binding pair member in a sample, by methods thatare well known in the art. Typically, the fluorescent labeled specificbinding pair member is added to a sample that contains, or is thought tocontain, the complementary specific binding pair member. Sufficient timeis allowed for the two members of the specific binding pair to form acomplex, the nature of said complex being dependent upon the type ofspecific binding pair utilized, but is typically characterized asnon-covalent (Van der Waals) interaction. After sufficient time haselapsed, the sample is observed for a colorimetric or fluorescent signalto indicate localization of the fluorescent conjugate. Detection of thecomplex is typically facilitated by washing or rinsing the sample toremove uncomplexed dye-conjugate.

While the resulting complex is detectable colorimetrically, usingambient light, typically the complex is detected by the fluorescenceproperties of the labeled specific binding pair member. Uponillumination, such as by an ultraviolet or visible wavelength emissionlamp, an arc lamp, a laser, or even sunlight or ordinary room light, thelabeled conjugates and specific binding pair complexes display intensevisible absorption as well as fluorescence emission. Selected equipmentthat is useful for illuminating the dye-conjugates of the inventionincludes, but is not limited to, hand-held ultraviolet lamps, mercuryarc lamps, xenon lamps, argon lasers, and YAG lasers. These illuminationsources are optionally integrated into laser scanners, fluorescencemicrotiter plate readers, standard or mini fluorometers, orchromatographic detectors. This colorimetric absorbance or fluorescenceemission is optionally detected by visual inspection, or by use of anyof the following: CCD cameras, video cameras, photographic film, laserscanning devices, fluorometers, photodiodes, quantum counters,epifluorescence microscopes, scanning microscopes, flow cytometers,fluorescence microtiter plate readers, or by means for amplifying thesignal such as photomultiplier tubes. Where the sample is examined usinga flow cytometer, a fluorescence microscope or a fluorometer, theinstrument is optionally used to distinguish and discriminate betweenthe dye-conjugate and a second fluorophore with detectably differentoptical properties, preferably, by distinguishing the fluorescenceresponse of the dye-conjugate from that of the second fluorophore. Wherethe sample is examined using a flow cytometer, examination of the sampleoptionally includes sorting the specific binding pair complex based onthe fluorescence response of the dye-conjugate.

It is also possible to utilize the dyes to label reactive sites such asoccur at the surface of cells, in cell membranes or in intracellularcompartments such as organelles, or in the cell's cytoplasm. Among thepreferred reagents are those that react selectively with intracellularglutathione (Example 10, Compound 8). Other fluorescent probes thatreact with intracellular glutathione have previously been described(U.S. Pat. No. 5,362,628 to Haugland et al, (1994); Copendingapplication HALOALKYL DERIVATIVES OF REPORTER MOLECULES USED TO ANALYZEMETABOLIC ACTIVITY IN CELLS, U.S. Pat. No. 5,576,424 to Haugland et al.(1996)).

In addition to their utility as a labeled specific binding pair member,conjugates prepared from any of the reactive dyes can also be used for avariety of other purposes, including any purposes that have beendescribed for conjugates of SSC. Primary applications include those inimmunofluorescence, fluorescence in situ hybridization, labeling ofreceptors with a low or high molecular weight fluorescent analog,conjugation to proteins or carbohydrates for microinjection into cells,and the tracing of labeled cells or polymers. The nucleotide conjugatesof the present invention are readily incorporated by DNA polymerase andcan be used for in situ hybridization or other techniques (Example 32).Also, the reactive dyes can be used to derivatize low molecular weightcompounds for their analysis by CZE, HPLC or other separationtechniques.

The examples below are given so as to illustrate the practice of thisinvention. They are not intended to limit or define the entire scope ofthis invention.

EXAMPLES Example 1 Preparation of 6-(4(or2)-(9-(2,3,6,7,12,13,16,17-octahydro-1H,5H-11H,15H-xantheno(2,3,4-ij:5,6,7-i′j′)diquinolizinyl-18-ium))-3(or5)-sulfo-1-phenylsulfonamido)hexanoic acid (Compound 1)

The following mixture of compounds is prepared:

To a solution of 0.60 g (3.30 mmol) of 6-aminohexanoic acid methyl esterand 1.0 mL of triethylamine in 50 mL of chloroform is added 1.50 g (2.40mmol) of SSC in small portions over a period of 10 minutes while thereaction mixture is stirred at 0° C. After the reaction mixture isstirred at room temperature 15 hours, it is washed with three 50 mLportions of water. The organic layer is separated, dried over anhydroussodium sulfate and concentrated under reduced pressure to give a darkpurple solid. This crude methyl ester derivative is purified bychromatography on silica gel with 2.5% methanol in chloroform as eluantto give 1.20 g (68%) of the methyl ester.

To a suspension of 0.80 g (1.09 mmol) of the above methyl ester in 15 mLof dioxane is added 25 mL of 6 M HCl dropwise over a period of 5minutes. After the reaction mixture is stirred at room temperature for18 hours, it is poured into 200 mL of water. The resulting solid iscollected by filtration and purified by chromatography on silica gelwith 15% methanol in chloroform as eluant. A dark purple solid (620 mg,79%) is obtained as a mixture of two isomers. TLC: R_(f)=0.54 (silicagel, 25% methanol in chloroform). ¹H NMR (DMSO-d₆): δ=8.40 (d, 1H, ArH),7.95-7.84 (m, 1H, ArH), 7.36 (d, 1H, ArH), 6.55 (d, 2H, ArH), 3.60-3.40(m, 8H, CH₂), 3.15-2.58 (m, 8H, CH₂), 2.20 (m, 8H, CH₂), 1.50-1.12 (m,6H, CH₂). Absorption maximum: 591 nm (ε=85,400 cm⁻¹M⁻¹) in pH 7.5phosphate buffer, 584 nm (e=94,400 cm⁻¹M⁻¹) in methanol, emissionmaximum: 610 nm in pH 7.5 phosphate buffer solution, 603 nm in methanol.

The two mixed isomers are resolved by HPLC under the followingconditions: Microsorb MV 86-800-D5, CN-Si column, 5μ, 4.6 mm id×25 cmlength (Rainin, Woburn, Mass.); eluted with acetontrile (1)/methanol(1)-0.1 M TEAA (triethylammonium acetate), pH 7.0, 5/95, flow rate 1mL/min, Isomer I; retention time of 22.40 minutes, absorption maximum:588 nm in pH 7.5 phosphate buffer, 581 nm in methanol, emission maximum:607 nm in pH 7.5 phosphate buffer, 599 nm in methanol. Isomer II;retention time of 23.68 minutes, absorption maximum: 590 nm in pH 7.5phosphate buffer, 583 nm in methanol, emission maximum: 612 nm in pH 7.5phosphate buffer, 602 nm in methanol.

Example 2 Preparation of a Succinimidyl Ester of Sulforhodamine 101(Compound 2)

The following mixture of compounds is prepared:

To a solution of 300 mg (0.42 mmol) of Compound 1 (Example 1) in 3 mL ofDMF is added 70 μL (0.50 mmol) of triethylamine, followed by addition of150 mg (0.50 mmol) of O-(N-succinimidyl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate. After the reaction mixture is stirred at roomtemperature for 1 hour, it is diluted with 50 mL of chloroform, washedwith water, dried over anhydrous sodium sulfate and concentrated underreduced pressure to give a solid. This solid is dissolved in 10 mL ofchloroform. The resulting solution is added dropwise into 100 mL ofether while stirring vigorously at room temperature. The resultingprecipitate is collected by filtration and dried under vacuum to give295 mg (87%) of a dark purple solid as mixed isomers. TLC: R_(f)=0.20(silica gel, 10% methanol in chloroform: ¹H NMR (DMSO-d₆): δ=8.39 (d,1H, ArH), 7.92-7.84 (m, 1H, ArH), 7.48-7.33 (m, 1H, ArH), 6.53 (d, 2H,ArH), 3.54-3.40 (m, 8H, CH₂), 3.13-2.57 (m, 8H, CH₂), 2.80 (s, 4H, CH₂),2.08-1.74 (m, 8H, CH₂), 1.65-1.12 (m 6H, CH₂). Absorption maximum: 591nm in pH 7.5 phosphate buffer, 582 nm in methanol; emission maximum: 611nm in pH 7.5 phosphate buffer, 603 nm in methanol.

The chemical reactivity of this succinimidyl ester is demonstrated bythin layer chromatography. n-Butylamine reacts with the succinimidylester to form a new product with R_(f)=0.27 (silica gel, 10% methanol inchloroform).

Example 3 Preparation of a Sulfosuccinimidyl Ester of Sulforhodamine 101(Compound 3)

The following mixture of compounds is prepared:

To a solution of 50 mg (0.07 mmol) of Compound 1 (Example 1) and 17 mg(0.08 mmol) of N-hydroxysulfosuccinimide, sodium salt in 2 mL of DMF isadded 15 μL (0.09 mmol) of N,N′-diisopropylcarbodiimide. After thereaction mixture is stirred at room temperature for 24 hours, it isadded to 20 mL of isopropyl alcohol. The resulting precipitate isremoved by filtration and the filtrate is concentrated under reducedpressure to remove most of the isopropanol. The residual DMF solution ispoured into 30 mL of ether to precipitate the product. The resultingprecipitate is collected by filtration to give 32 mg (50%) of thesulfosuccinimidyl ester as a dark purple solid. TLC: R_(f)=0.20 (silicagel, 25% methanol in chloroform). ¹H NMR (DMSO-d₆): δ=8.40 (d, 1H, ArH),7.90-7.84 (m, 1H, ArH), 7.48-7.33 (m, 1H, ArH), 6.53 (d, 2H, ArH),3.95-3.90 (m, 1H, CH), 3.77-3.70 (m, 2H, CH₂), 3.55-1.12 (s, 34H, CH₂).Absorption maximum: 591 nm in pH 7.5 phosphate buffer, 584 nm inmethanol; emission maximum: 611 nm in pH 7.5 phosphate buffer, 604 nm inmethanol.

The reactivity of this sulfosuccinimidyl ester is demonstrated by thinlayer chromatography. n-Butylamine reacts with this sulfosuccinimidylester to form a new product with R_(f)=0.57 (silica gel, 25% methanol inchloroform).

Example 4 Preparation of a Nucleotide Conjugate of Sulforhodamine 101

To a solution of 3 mg of5-(3-aminoallyl)-2′-deoxyuridine-5′-triphosphate, ammonium salt (SigmaChemical) in 300 μL of water is added a solution of 4 mg of Compound 2(Example 2) in 200 μL of DMF, followed by addition of 5 μL oftriethylamine. After the mixture is stirred at room temperature for 3hours, it is purified by chromatography over lipophilic SEPHADEX resinusing water for elution. The desired fractions are combined andlyophilized to give 3 mg of the fluorescent nucleotide conjugate as adark purple solid.

Example 5 Preparation of an Oligonucleotide Conjugate of Sulforhodamine101

A sample of 500 μg of a 5′-amine modified, 24-base M13 primer sequenceis dissolved in 220 μL of 0.1 M borated sodium bicarbonate pH 8.5aqueous buffer in the microcentrifuge tube. To this oligonucleotidesolution is added a solution of 1 mg of Compound 3 (Example 3) in 35 μLof DMF. The reaction mixture is shaken by hand for a few minutes andallowed to stand at room temperature for 16 hours. To the mixture isadded 15 μL of 5 M NaCl and 3 volumes of cold 100% ethanol. Theresulting mixture is incubated at −20° C. for 30-60 minutes, and thenmicrocentrifuged for 15-30 minutes at 4° C. (5,000-10,000 g). Aftermicrocentrifugation, the ethanol supernate is decanted, and the pelletis resuspended in 100 μL H₂O. The labeled oligonucleotide is thenpurified by HPLC on a 220 mm×10 mm 300 Å C8 reverse phase column (RaininInstrument Co., Woburn, Mass.) using the following gradient: SolventA—0.1 M TEAA (pH˜7), Solvent B—acetonitrile. Ramp Solvent B from 15% to60% over 30 minutes. Detection is accomplished using a Waters 490 dualwavelength UV-Vis detector monitoring 254 nm and 590 nm. The desiredpeak is collected and evaporated to give approximately 200 μg of thefluorescent oligonucleotide.

A comparison of labeling efficiency of both SSC and Compound 2 whenconjugating an M13 primer using the procedure given above shows thatCompound 2 labels the oligonucleotide approximately two-fold moreefficiently than SSC, as determined by comparing the ratio of theintegrated absorbance of the product peaks to that of the unlabeledoligonucleotide during HPLC.

Example 6 Preparation of a Phalloidin Conjugate (Compound 4)

To a solution of 3 mg of aminophalloidin p-toluenesulfonate and 4 mg ofCompound 2 (Example 2) in 300 μL of DMF is added 5 μL of triethylamineand the mixture is stirred at room temperature for 1 hour. To thereaction mixture is added 7 mL of ether and the resulting precipitate iscollected by centrifugation. The crude product is purified bychromatography over lipophilic SEPHADEX resin using water for elution.The desired fractions are combined and lyophilized to give 4 mg of adark purple solid as a fluorescent phalloidin conjugate.

Example 7 Preparation of an Iodoacetamide-modified Sulforhodamine 101,Compound 5

The following mixture of compounds is prepared:

To a suspension of 50 mg (0.07 mmol) of sulforhodamine 101 sulfonylcadaverine in 10 mL of chloroform is added 15 μL (0.09 mmol) ofN,N-diisopropylethylamine, followed by addition of 20 mg (0.07 mmol) ofsuccinimidyl iodoacetamide and the mixture is stirred at roomtemperature for 18 hours. The reaction mixture is then diluted with 50mL of chloroform, washed with water (3×50 mL), dried over anhydroussodium sulfate and concentrated under reduced pressure to give a crudeproduct. The product is purified by chromatography on silica gel with 5%methanol in chloroform as eluant to give 14 mg (23%) of theiodoacetamide as a dark purple solid. TLC: R_(f)=0.21 (silica gel, 10%methanol in chloroform). ¹H NMR (DMSO-d₆): δ=8.38 (d, 1H, ArH),7.90-7.80 (m, 1H, ArH), 7.38-7.35 (m, 1H, ArH), 6.50 (d, 2H, ArH), 3.60(s, 2H, CH₂I), 3.58-3.40 (m, 8H, CH₂), 3.05 (m, 6H, CH₂), 2.68-2.54 (m,6H, CH₂), 2.08-1.98 (m, 4H, CH₂), 1.90-1.78 (m, 4H, CH₂), 1.45-1.12 (m,6H, CH₂). Absorption maximum: 591 nm in pH 7.5 phosphate buffer, 584 nmin methanol; emission maximum: 613 nm in pH 7.5 phosphate buffer, 603 nmin methanol.

The chemical reactivity of this iodoacetamide is demonstrated by thinlayer chromatography. N-Acetylcysteine reacts with this iodoacetamide toform a new product with R_(f)=0.08 (silica gel, 25% methanol inchloroform).

Example 8 Preparation of Bromoacetamide Modified Sulforhodamine 101,Compound 6

The following mixture of compounds is prepared:

To a suspension of 50 mg (0.07 mmol) of sulforhodamine 101 sulfonylcadaverine in 10 mL of chloroform is added 15 μL (0.09 mmol) ofN,N-diisopropylethylamine, followed by addition of 7 μL (0.08 mmol) ofbromoacetyl bromide and the reaction mixture is stirred at roomtemperature for 5 hours. It is then diluted with 50 mL of chloroform,washed with water (3×50 mL), dried over anhydrous sodium sulfate andconcentrated under reduced pressure to give a crude product. The productis purified by chromatography on silica gel with 5%. methanol inchloroform as eluant to give 41 mg (73%) of the bromoacetamide as a darkpurple solid. TLC: R_(f)=0.20 (silica gel, 10% methanol in chloroform).¹H NMR (DMSO-d₆): δ=8.38 (d, 1H, ArH), 7.91-7.80 (m, 1H, ArH), 7.40-7.35(m, 1H, ArH), 6.50 (d, 2H, ArH), 3.80 (s, 2H, CH₂Br), 3.60-3.40 (m, 8H,CH₂), 3.06-2.83 (m, 6H, CH₂), 2.67-2.53 (m, 6H, CH₂), 2.09-1.97 (m, 4H,CH₂), 1.90-1.78 (m, 4H, CH₂), 1.44-1.12 (m, 6H, CH₂). Absorptionmaximum: 591 nm in pH 7.5 phosphate buffer, 583 nm in methanol; emissionmaximum: 610 nm in pH 7.5 phosphate buffer, 603 nm in methanol.

The chemical reactivity of this bromoacetamide is demonstrated by thinlayer chromatography. N-acetyl cysteine reacts with this bromoacetamideto form a new product with R_(f)=0.08 (silica gel, 25% methanol inchloroform).

Example 9 Preparation of a Maleimide-modified Sulforhodamine 101,Compound 7

The following mixture of compounds is prepared:

To a solution of 50 mg (0.07 mmol) of sulforhodamine 101 sulfonylcadaverine and 25 μL (0.14 mmol) of N,N-diisopropylethylamine in 5 mL ofchloroform is added 10 mg (0.10 mmol) of maleic anhydride. After thereaction mixture is stirred at room temperature for 5 hours, it isconcentrated to a volume of 1 mL under reduced pressure. The residualsolution is poured into 10 mL of ether. The resulting precipitate iscollected by filtration to give 43 mg of the maleamic acid intermediateas a dark purple solid.

A suspension of this intermediate and 10 mg of sodium acetate in 2 mL ofacetic anhydride is heated at 90° C. After 30 minutes the reactionmixture is concentrated under reduced pressure. The residue is dilutedwith 50 mL of chloroform, washed with water (3×50 mL), dried overanhydrous sodium sulfate and concentrated under reduced pressure to givea crude product. The crude product is purified by chromatography onsilica gel with 5% methanol in chloroform as eluant to give 18 mg (32%)of a maleimide as mixed isomers. For analytical purposes, small portionsof this sample are further purified by preparative thin layerchromatography on silica gel using 10% methanol in chloroform as eluant.Two isomers are separated. Isomer 1; TLC: R_(f)=0.15 (silica gel, 10%methanol in chloroform); Absorption maximum: 590 nm in pH 7.5 phosphatebuffer, emission maximum: 610 nm in pH 7.5 phosphate buffer; ¹H NMR(DMSO-d₆): δ=8.42 (s, 1H, ArH), 8.06 (d, 1H, ArH), 7.42 (d, 1H, ArH),7.01 (d, 2H, —CH═CH—), 6.50 (s, 2H, ArH), 3.50-1.18 (m, 34H, CH₂), 2.37(s, 3H, CH₃). Isomer 2; TLC: R_(f)=0.13 (silica gel, 10% methanol inchloroform); Absorption maximum: 601 nm in pH 7.5 phosphate buffer;emission maximum: 619 nm in pH 7.5 phosphate buffer; ¹H NMR (DMSO-d₆):δ=8.41 (s, 1H, ArH), 8.08 (d, 1H, ArH), 7.44 (d, 1H, ArH), 7.01 (d, 2H,—CH═CH—), 6.44 (s, 2H, ArH), 3.58-1.19 (m, 34H, CH₂), 1.97 (s, 3H, CH₃).

The chemical reactivity of this maleimide is demonstrated by thin layerchromatography. N-acetyl cysteine reacts with this maleimide to form anew product with R_(f)=0.10 (silica gel, 25% methanol in chloroform).

Example 10 Preparation of a Chloromethylbenzamide ModifiedSulforhodamine 101, Compound 8

The following mixture of compounds is prepared:

To a solution of 100 mg (0.14 mmol) of sulforhodamine 101 sulfonylcadaverine and 45 μL (0.32 mmol) of triethylamine in 30 mL of chloroformis added 20 mg (0.16 mmol) of 4-(chloromethyl)benzoyl chloride. Afterthe reaction mixture is stirred at room temperature for 3 hours, it iswashed with water (3×30 mL), dried over anhydrous sodium sulfate andconcentrated under reduced pressure to give a crude product. This crudeproduct is purified by chromatography on silica gel with 5% methanol inchloroform as eluant to give 97 mg (79%) of a pure product as mixedisomers. ¹H NMR (DMSO-d₆): δ=8.40 (d, 1H, ArH), 7.98-7.90 (m, 1H, ArH),7.78 (d, 2H, ArH), 7.42 (d, 2H, ArH), 7.33 (d, 1H, ArH), 6.50 (s, 2H,ArH), 4.80 (s, 2H, CH₂Cl), 3.54-1.21 (m, 34H, CH₂). For analyticalpurposes, small portions of this product are further separated bypreparative thin layer chromatography on silica gel using 10% methanolin chloroform as eluant. Two isomers are separated. Isomer 1: R_(f)=0.18(silica gel, 10% methanol in chloroform), absorption maximum: 589 nm inpH 7.5 phosphate buffer, emission maximum 608 nm in pH 7.5 phosphatebuffer. Isomer 2: R_(f)=0.16 (silica gel, 10% methanol in chloroform),absorption maximum: 591 nm in pH 7.5 phosphate buffer; emission maximum:612 nm in pH 7.5 phosphate buffer.

Example 11 Preparation of a Phospholipid Modified Sulforhodamine 101,Compound 9

The following mixture of compounds is prepared:

To a solution of 200 mg (0.24 mmol) of Compound 2 (Example 2) in 50 mLof chloroform is added 35 μL (0.25 mmol) of triethylamine, followed byaddition of 170 mg (0.24 mmol) of1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine. After the reactionmixture is stirred at room temperature for 24 hours, it is concentratedunder reduced pressure. The resulting crude product is purified bychromatography on silica gel with 15% methanol in chloroform as eluantto give 290 mg (79%) of the product as mixed isomers. TLC: R_(f)=0.33(silica gel, 25% methanol in chloroform). ¹H NMR: (DMSO-d₆): δ=8.49 (d,1H, ArH), 7.97-7.40 (m, 1H, Arh), 7.32 (d, 1H, ArH), 6.49 (d, 2H, ArH),5.14-5.07 (m, 1H, CH), 4.34-1.14 (m, 104H, CH₂), 1.06 (t, 9H, CH₃), 0.85(t, 6H, CH₃). TLC: absorption maximum: 583 nm in methanol; emissionmaximum: 602 nm in methanol.

Example 12 Preparation of an Acylazide Derivative of Sulforhodamine 101,Compound 10

The following mixture of compounds is prepared:

To a solution of 50 mg (0.07 mmol) of Compound 1 (Example 1) and 20 μL(0.14 mmol) of triethylamine in 5 mL of 1,2-dichloroethane is added 20μL (0.09 mmol) of diphenylphosphoryl azide. After stirring at roomtemperature for 18 hours, the reaction mixture is subjected to columnchromatography on silica gel with 5% methanol in chloroform as eluant.From the desired combined fractions 29 mg (60%) of a product as mixedisomers is obtained.

The reactive isocyanate derivative of sulforhodamine 101 is preparedfrom Compound 10 by a Curtius rearrangement of the acyl azide, followedby reaction with an appropriate alcohol. For example, a solution of 10mg of acyl azide and 5 mg of cholesterol in 5 mL of dry1,2-dichloroethane is heated under reflux in a stream of N₂ for 2 hours.After the reaction mixture is cooled to room temperature, thin layerchromatography on silica gel is analyzed to show a new spot for afluorescent cholesterol conjugate (Compound 11).

Example 13 Preparation of an Isothiocyanate Derivative ofSulforhodamine, Compound 12

The following mixture of compounds is prepared:

To a solution of 50 mg (0.07 mmol) of sulforhodamine 101 sulfonylcadaverine in 10 mL of dichloromethane is added 17 mg (0.07 mmol) of1,1′-thiocarbonyldi-2(1H)-pyridone. After stirring at room temperaturefor 1 hour, the reaction mixture is washed with water (2×10 mL), driedover anhydrous sodium sulfate and concentrated under reduced pressure togive 41 mg (80%) of a product as a dark purple solid. Thisisothiocyanate reacts with n-butylamine to give a new spot on thin layerchromatography.

Example 14 Preparation of a Dichlorotriazine Derivative ofSulforhodamine 101, Compound 13

The following mixture of compounds is prepared:

To a suspension of 15 mg (0.08 mmol) of cyanuric chloride in 2 mL ofchloroform is added a solution of 50 mg (0.07 mmol) of sulforhodamine101 sulfonyl cadaverine in 2 mL of methanol while the mixture is stirredin an ice-water bath. After stirring at an ice-water bath temperaturefor 1 hour, the reaction mixture is poured into 15 mL of ether. Theresulting precipitate is collected by filtration to give 45 mg (70%) ofa desired product as a dark purple solid.

Example 15 Preparation of a Maleimide-modified Sulforhodamine 101,Compound 14

The following mixture of compounds is prepared:

To a solution of 55 mg (0.08 mmol) of sulforhodamine 101 sulfonylcadaverine and 12 μL (0.08 mmol) of triethylamine in 15 mL of chloroformis added 20 mg (0.08 mmol) of succinimidyl maleimidylacetate. After thereaction mixture is stirred at room temperature for 3 hours, it iswashed with water (2×15 mL), dried over anhydrous sodium sulfate andconcentrated under reduced pressure to give a crude product. This crudeproduct is purified by chromatography on silica gel with 3% methanol inchloroform as eluant to give 35 mg (53%) of the pure product as amixture of isomers: R_(f)=0.20 (silica gel, 10% methanol in chloroform),absorption maximum: 583 nm in methanol; emission maximum: 602 nm inmethanol.

Example 16 Preparation of an Azide-modified Sulforhodamine 101, Compound15

The following mixture of compounds is prepared:

To a solution of 25 mg (0.04 mmol) of sulforhodamine 101 sulfonylcadaverine and 6 μL (0.04 mmol) of triethylamine in 10 mL of chloroformis added 14 mg (0.04 mmol) of 4-azido-2,3,5,6-tetrafluorobenzoic acid,succinimidyl ester. After the reaction mixture is stirred at roomtemperature for 4 hours, it is washed with water (2×10 mL), dried overanhydrous sodium sulfate and concentrated under reduced pressure to givea crude product. This crude product is purified by chromatography onsilica gel with 3% methanol in chloroform as eluant to give 21 mg (65%)of the desired product as a dark purple solid.

Example 17 Preparation of a Drug Conjugate of Sulforhodamine 101,Compound 16

A fluorescent dopamine D₂ antagonist is prepared as follows:

To a solution of 10 mg (0.02 mmol) of N-(p-aminophenethyl)spiperone,which is prepared as described in Amlaiky, et al., FEBS LETT, 176, 436(1984), and 10 μL (0.06 mol) of N,N-diisopropylethylamine in 1 mL of DMFis added 15 mg (0.02 mmol) of Compound 12 (Example 13). After thereaction mixture is stirred at room temperature for 3 hours, it ispoured into 5 mL of ether. The resulting precipitate is collected bycentrifugation. This crude product is purified by chromatography onsilica gel using 10% methanol in chloroform to give 21 mg (85%) of apure product as a dark purple solid.

Example 18 Preparation of a Peptide Conjugate of Sulforhodamine 101,Compound 17

To a suspension of 5 mg of an N-formyl modified hexapeptide (a potentchemoattractant for human neutrophils, Niedel et al. SCIENCE, 205, 1412(1979) in 500 μL of DMF is added 3 μL of triethylamine followed by theaddition of 5 mg of Compound 2 (Example 2) and the whole reactionmixture is stirred at room temperature for 1 hour. The reaction mixtureis purified by chromatography over lipophilic SEPHADEX resin using waterfor elution. The desired fractions are combined and lyophilized to give5 mg of the fluorescent hexapeptide.

Example 19 Protein Conjugates of Sulforhodamine 101

Protein conjugates of sulforhodamine 101 are prepared using Compound 2(Example 2). The degree of substitution achieved on the selectedproteins (bovine serum albumin (BSA), goat anti-mouse IgG (GAM) orstreptavidin (STR)) is then determined.

A fresh solution of the desired protein is prepared that is 10 mgprotein/mL in 0.1 M sodium bicarbonate. The labeling reagent (Compound2) is dissolved in DMF to give a concentration of 10 mg dye/mL.Predetermined amounts of the labeling reagent in DMF are slowly added tothe protein solution with stirring. A molar ratio of 10 equivalents dyeto equivalent of protein is typical, though the optimal amount varieswith particular labeling reagent and protein being labeled. The reactionmixture is incubated at room temperature for one hour. The dye-proteinconjugate is separated from free unreacted reagent by gel filtration ona CELLUFINE GH-25 column equilibrated in PBS. The initial,protein-containing colored band is collected from the column, and thedegree of substitution is determined by measuring the absorbance of theconjugate at 595 nm, and calculating the degree of substitution using anextinction coefficient of 80,000 cm⁻¹M⁻¹ for the dye.

Dye conjugates are similarly prepared using SSC excepting that theprotein solution is maintained at pH 9, and the labeling reaction isconducted at ice bath temperatures to minimize hydrolysis of thelabeling reagent.

The degree of substitution achieved with each labeling reagent on theselected protein is shown in the following table:

SSC Compound 2 Dye:Protein Degree of Dye:Protein Degree of Protein RatioSubstitution Ratio Substitution bovine serum 15 3.2 15 4.5 albumin (BSA)goat 2.5 0.57 2.5 1.29 anti-mouse (GAM) 5.5 1.0 5.5 2.54 8.0 1.3 8.03.27 streptavidin 1 0.066 1 0.76 (STR) 2.5 1.2 2.5 1.6 5.5 2.4 5.5 4.48.0 3.0 8.0 5.6 10 3.9 10 6.0 20 4.9 20 6.0 30 5.1 30 6.9

As shown above, the use of the reactive dye of the present invention asa labeling reagents results in a greater degree of dye incorporationinto the protein at similar molar ratios of reactive dye to protein.

Example 20 Preparation of an Energy-transfer Conjugate

Conjugates of R-Phycoerythrin (R-PE) with Compound 2 (Example 2) or SSCare prepared as described in Example 19 at dye/protein ratios of 10, 20and 30. The purified conjugates are then excited at 488 nm, and theemission spectra are recorded. The R-PE conjugate of Compound 2(prepared with a dye/protein ratio of 20) exhibits significant energytransfer with little fluorescence emission at the wavelengths expectedfor the R-PE fluorophore, while the R-PE conjugate of SSC exhibitsrelatively poor energy transfer.

The R-PE conjugate of Compound 2 possesses significant utility as asecond color probe used in conjunction with a first green oryellow-green emitting dye. Although the R-PE conjugate is successfullyexcited at the same wavelengths as typical green dyes, its emission isshifted to wavelengths appropriate for sulforhodamine 101.

Example 21 Hydrolytic Stability of Compound 2

Compound 2 and SSC are separately dissolved in solutions that are 90%DMF and 10% aqueous sodium bicarbonate. Each solution is analyzed usingthin layer chromatography (TLC) as follows: An aliquot from each sampleis diluted 1:100 in methanol and 1 μL of the resulting solution isspotted on a silica gel TLC plate and developed in 15% methanol inchloroform. The SSC reagent is so labile that it does not withstand theTLC conditions and decomposes immediately upon application to the TLCplate. However, only 10-20% of Compound 2 appears to decompose evenafter two hours of incubation in the presence of water.

Example 22 The Effect of Hydrolytic Stability on Protein LabelingEfficiency

Compound 2 and SSC are separately dissolved at room temperature insolutions that are 25% DMF and 75% aqueous sodium bicarbonate. Afterperiods of 1, 5, 10, 20, 30, and 60 minutes, the dye solutions are usedto label goat IgG protein at a dye/protein ratio of 10 and a proteinconcentration of 10 mg/mL in 100 mM sodium bicarbonate. The resultingprotein conjugates are purified as described in Example 19. Theresulting degrees of substitution are shown below:

Time Degree of Substitution (minutes) SSC Compound 2 1 0.30 4.09 5 —3.64 10 — 3.38 20 — 2.96 30 — 3.00 60 — 2.78

Due to the high hydrolytic instability of SSC, no protein conjugate isformed when the reagent is exposed to water at room temperature for aslittle as five minutes. In contrast, the labeling of proteins usingCompound 2 of the present invention results in adequate labeling evenafter 1 hour in aqueous solution.

Example 23 Total Fluorescence of Selected Dye Conjugates as a Functionof Degree of Substitution

The total fluorescence of selected conjugates of the dyes of the presentinvention is plotted against the degree of substitution of theconjugate. Total fluorescence is the product of the degree ofsubstitution and the quantum yield, relative to a common standard (inthis case, sulforhodamine 101). The degree of substitution is determinedas described earlier (Example 19). The results of this calculation forstreptavidin conjugates of SSC, Compound 2, and XRITC show the optimaldegree of substitution for each conjugate and the relative fluorescenceintensity of conjugates made with each dye, as plotted graphically inFIG. 5. As shown, as the degree of substitution increases, the totalfluorescence increases, until the point at which the quantum yieldbegins to decrease due to crowding of the dye molecules and theresultant fluorescence quenching, thereby canceling the effect ofadditional dye substitution.

Example 24 Utility of Protein Conjugates as Immunoreagents

The succinimidyl ester derivative Compound 2 is used to prepareconjugates of streptavidin that are further utilized in immunochemistryexperiments. In particular, the efficacy of the streptavidin conjugateof Compound 2 (prepared as described in Example 19) is tested inparallel with the streptavidin conjugate of SSC. The comparison isperformed using a test to detect antinuclear antibodies commerciallyavailable from INOVA Diagnostics Inc. (San Diego, Calif.). Thecommercial assay consists of a series of fixed cells on slides, and anautoimmune serum against cell nuclei. The two streptavidin conjugates ofCompound 2 exhibit degrees of substitution of 2.2 and 4.2 moles of dyeper mole of protein, respectively, while the two streptavidin conjugatesof SSC exhibit degrees of substitution of 2.4 and 3.9 dyes per mole. Thecell nuclei are treated with either positive serum or negative serum (asa control), and are then developed with biotinylated protein A. Each ofthe streptavidin conjugates above is then used to label the treatedcells at a concentration of 5 μg/mL. Each of the Compound 2 streptavidinconjugates yield lower fluorescence background and brighter nuclearstaining than the corresponding SSC-streptavidins (approximatelytwo-fold more fluorescent, as measured using a fluorescent microscopecoupled to a Photometrics Star-1 cooled CCD camera for quantitativedigital imaging).

Example 25 Labeling Efficiency of Thiol-reactive Dyes

The thiol-reactive dyes Compound 5 (iodoacetamide-modified), Compound 6(bromoacetamide-modified) and Compound 7 (maleimide-modified) are testedfor labeling efficiency by reacting the dyes with b-galactosidase, anenzyme with more then 10 free thiol groups per molecule. Each dye isdissolved at a concentration of 10 mg/mL in DMF and added to 3 mg of theprotein dissolved at 10 mg/mL in 0.1 M phosphate, 0.1 M NaCl pH 7.5. Thedyes are added at molar ratios of 10 for the maleimide and 20 and 40 forboth the bromoacetamide and the iodoacetamide, respectively. Thereaction is continued for 1 hour at room temperature under argon, andthe conjugates are purified using column chromatography. The degree ofsubstitution obtained using each of the three compounds is shown below:

Reactive Dye: Protein Ratio Reactive Dye 10 20 40 Compound 5 — 4.91 5.14Compound 6 — 4.20 4.70 Compound 7 7.63 — —

As demonstrated by the above data, the thiol-reactive dyes are usefulfor labeling proteins which have free thiol groups.

Example 26 Solubility of Compound 2

The solubility of Compound 3 is determined by dissolving the reactivedye in 0.1 M aqueous sodium bicarbonate, in the absence of organicsolvents at room temperature. This sulfosuccinimidyl ester possesses asolubility of about 10 mg/mL, when dissolution is facilitated by the useof sonication.

Example 27 Preparation of a Dextran Conjugate of Sulforhodamine 101

The dichlorotriazine derivative (Compound 13) is used to label thehydroxyl groups of a polysaccharide with sulforhodamine 101fluorophores. A 40,000 MW dextran (50 mg) is dissolved in 2.5 mL of 0.2M sodium carbonate buffer (pH 9.5) and the resulting solution is heatedto 50° C. in a temperature controlled bath. A solution of 20 mg ofCompound 5 in 1 mL DMSO is added to the dextran solution with stirring.The reaction is continued for 6 hours, maintaining the pH at 9.5-10.0 bythe addition of aliquots of 1 M NaOH. The dye-dextran conjugate is thenpurified on a SEPHADEX G-50 resin chromatographic column that has beenequilibrated with 30 mM ammonium acetate. The first colored band toelute is collected, and the dextran solution is lyophilized. The degreeof labeling is determined as in Example 19. The degree of substitutionobtained is 5 dyes/40,000 daltons of dextran.

Example 28 Preparation of an Aminodextran Conjugate of Sulforhodamine101

A sample of aminodextran (50 mg) having an average molecular weight of70,000 and derivatized with an average of 13 amino groups, is dissolvedin 0.1 M sodium bicarbonate to give a concentration of 10 mg/mL. Asolution of Compound 2 in DMF having a concentration of 10 mg/mL isadded to the dextran solution in an amount to give a dye/protein ratioof 12. After stirring at room temperature, the conjugated dextrans arepurified by gel filtration using SEPHADEX G-50 resin in water. Thedextran solution is lyophilized, and the degree of substitution of thedextran is determined as described in Example 19. The dextran-conjugateexhibits a DOS of 6 dyes/70,000 daltons of dextran.

Aminodextran having an average molecular weight of 40,000 andderivatized with 7 amino groups is conjugated exactly analogously, usinga dye/protein ratio of 8. The resulting dextran-conjugate exhibits a DOSof 3 dyes/40,000 daltons of dextran.

Example 29 Labeling Actin in Cells Using a Phalloidin Conjugate ofSulforhodamine 101

Mammalian cells are grown on coverslips according to standard tissueculture procedures. After two days in culture, the growth medium isremoved and the cells are rinsed twice with warm Hanks Balanced SaltSolution (HBSS; 0.14 g/L CaCl₂, 0.40 g/L KCl, 0.06 g/L KH₂PO₄, 0.10 g/LMgCl₂.6H₂O, 0.10 g/L MgSO₄.7H₂O, 8.0 g/L NaCl, 0.35 g/L NaHCO₃, 0.48 g/LNa₂HPO₄, 1 g/L D-glucose). Cells are then fixed in 3.7% formaldehydediluted into HBSS for 10 minutes at room temperature. Cells are rinsedin phosphate buffered saline (PBS; 0.20 g/L KCl, 0.20 g/L KH₂PO₄, 8 g/lNaCl, 1.15 g/L Na₂HPO₄), and permeabilized in ice cold acetone for 10minutes. The cells are then rehydrated in PBS for 10 minutes, andstained with a 165 nM solution of Compound 4 in PBS. The stained cellsare then rinsed twice with PBS, mounted in the mounting medium ofchoice, and viewed using a standard filter set used for SSC conjugateson a fluorescence microscope. The staining of F-actin filaments usingCompound 4 is consistent with that of similar, shorter wavelengthphallotoxin conjugates.

Example 30 Labeling Actin in Cells Using a Phalloidin Conjugate ofSulforhodamine in Conjunction with a Labeled Antibody

Cells are grown, fixed and permeabilized as described in Example 29.After a 10 minutes rehydration in PBS, cells are blocked in a solutionof 1% bovine serum albumin/1% normal goat serum/0.1% TWEEN-20 in PBS for30 minutes. Monoclonal anti-tubulin antibody is diluted into theblocking buffer at a concentration of 2 μg/mL (Boehringer Mannheim,Indianapolis, Ind.) and a 100 μL volume per coverslip is incubated withthe cells for 1 hour. After rinsing in PBS, fluorescein goat anti-mouseantibody is diluted to 10 μg/mL and incubated with the cells for 30minutes. After rinsing in PBS, cells are then incubated with a 165 nMsolution of Compound 4 diluted in PBS for 30 minutes. Cells are rinsed afinal time in PBS, mounted in the mounting medium of choice, and viewedthrough either a multiband filter, or through a long-wavelength filterand a fluorescein filter with a standard fluorescence microscope.Simultaneous staining of both F-actin and tubulin filaments isconsistent with staining by the individual probes.

Alternatively, cells may be prepared according to other methods offixation and permeabilization.

Example 31 Fluorescence of Labeled Nucleotides

Two fluorescent conjugates of deoxyuridine-5′-triphosphate are preparedas in Example 4, only using5-(3-amino-1-propynyl)-2′-deoxyuridine-5′-triphosphate in place of5-(3-aminoallyl)-2′-deoxyuridine-5′-triphosphate (as described in Hobbs,Jr. et al, supra). One conjugate is prepared using Compound 2, the otheris prepared using SSC.

The two conjugates are separately dissolved in pH 8 buffer, and theoptical density of each solution is adjusted to 0.03 when measured at560 nm. Each solution is then excited at 560 nm and the resultingfluorescence emission is recorded from 565 nm to 750 nm. The results aredepicted in FIG. 6. The compound 2-dUTP conjugate exhibits a narroweremission spectrum than the SSC-dUTP conjugate. In addition, the SSC-dUTPconjugate displays a fluorescence yield that is only 75% of the measuredfluorescence yield for the Compound 2-dUTP conjugate.

Example 32 Preparing a DNA Hybridization Probe Using FluorescentNucleotide Conjugates

For each labeling reaction, a microfuge tube containing about 1 μg of a˜700 bp Hind III—Bgl II fragment of the E. coli lacZ structural gene isheated for about 10 minutes at 95° C. to fully separate the strands. TheDNA is immediately cooled on ice, to prevent the strands fromreannealing. To the DNA mixture on ice is added 2 μL of a 2 mg/mLmixture of random sequence hexanucleotides, in 0.5 M Tris-HCl, pH 7.2,0.1 M MgCl₂, 1 mM dithiothreitol; 2 μL of a dNTP labeling mixture (1 mMdATP, 1 mM dGTP, 1 mM dCTP, 0.65 mM dTTP and 0.35 mM either SSC labeleddUTP or Compound 2 labeled dUTP, as prepared in Example 4 or Example31). Sterile distilled and deionized water is added to the samples tobring the total volume of each to 19 μL. A 1 μL volume of Klenow DNApolymerase (2 units/μL) is added carefully to the samples and they aremixed by pipetting up and down repeatedly. The samples are incubated forone hour at 37° C. The reactions are stopped by adding 2 μL of 0.2 MEDTA, pH 8.0. The labeled DNA is precipitated by addition of 2.5 μL of 4M LiCl and 75 μL prechilled (−20° C.) 100% ethanol and mixing well.Precipitation is allowed to continue for 2 hours at −20° C. and thenucleic acids are then recovered by centrifugation at 5000 rpm in amicrofuge. The pellets are washed briefly with cold 70% ethanol, thenwith cold 100% ethanol. The pellets are dried briefly and dissolved in10 mM Tris-HCl, pH 8.0, 1 mM EDTA. A portion consisting of {fraction(1/10)} to ½ of each sample is analyzed by gel electrophoresis on a 1%agarose minigel under standard conditions. Both the SSC labeled dUTP andthe Compound 2 labeled dUTP give rise to clearly visible labeled DNAproducts that exhibit bright red fluorescence when visualized usingultraviolet trans or epi-illumination. The labeled DNA products aresuitable for in situ hybridization experiments for the detection of RNAor DNA associated with the E. coli lacZ gene in cells or tissues.

It is to be understood that, while the foregoing invention has beendescribed in detail by way of illustration and example, numerousmodifications, substitutions, and alterations are possible withoutdeparting from the spirit and scope of the invention as described in thefollowing claims.

What is claimed is:
 1. A reactive dye having the formula

or the formula

wherein R² is H, C₁-C₆ alkyl or C₁-C₆ acyl; and n=2 to 8; and R is ahalomethylbenzamidyl, maleimidyl,(3,5-dichloro-2,4,6-triazin-1-yl)amino, isocyanato or an isothiocyanato;or n=1 to 7; and R is a carboxylic acid, an alkali or alkaline earthmetal salt of a carboxylic acid, or R is —(C═O)—R_(x), where R_(x) is achloride, fluoride or azide; or R_(x) is selected so as to make R ahydroxybenzotriazolyl ester, a succinimidyl ester, a sulfosuccinimidylester, an alkali or alkaline earth metal salt of a sulfosuccinimidylester, a symmetric anhydride that links two sulforhodamine 101fluorophores, a mixed anhydride of a sulforhodamine 101 fluorophore anda chloroformate having 2-8 carbons, a mixed anhydride of asulforhodamine 101 fluorophore and a carboxylic acid or perfluorinatedcarboxylic acid having 2-8 carbons, a mixed anhydride of asulforhodamine 101 fluorophore and a sulfonic or fluorinated sulfonicacid having 1-8 carbons, or an ester of a phenol or a naphthol that isfurther substituted one or more times by nitro, sulfo, carboxy, alkalior alkaline earth metal salt of sulfo or carboxy, cyano, fluoro, chloro,or trifluoromethyl; or R_(x) is selected so as to make R a carboxylicacid activated by a carbodiimide having 2-14 carbons.
 2. A reactive dye,as claimed in claim 1, wherein n=2-8, and R is a halomethylbenzamido,maleimido, maleimidyl benzamido, maleimidyl alkylamido, azidobenzamido,azidoperfluorobenzamido, (3,5-dichloro-2,4,6-triazin-1-yl)amino,isocyanato or an isothiocyanato.
 3. A reactive dye, as claimed in claim2, wherein n=5 or
 6. 4. A reactive dye, as claimed in claim 2, wherein Ris maleimido or a halomethylbenzamido.
 5. A reactive dye, as claimed inclaim 2, wherein R is maleimido.
 6. A reactive dye, as claimed in claim5, having the formula


7. A reactive dye, as claimed in claim 1, wherein n=1-7 and R is acarboxylic acid, an alkali or alkaline earth metal salt of a carboxylicacid, or R is —(C═O)—R_(x), where R_(x) is a chloride, fluoride orazide; or R_(x) is selected so as to make R a hydroxybenzotriazolylester, a succinimidyl ester, a sulfosuccinimidyl ester, an alkali oralkaline earth metal salt of a sulfosuccinimidyl ester, a symmetricanhydride that links two sulforhodamine 101 fluorophores, a mixedanhydride of a sulforhodamine 101 fluorophore and a chloroformate having2-8 carbons, a mixed anhydride of a sulforhodamine 101 fluorophore and acarboxylic acid or perfluorinated carboxylic acid having 2-8 carbons, amixed anhydride of a sulforhodamine 101 fluorophore and a sulfonic orfluorinated sulfonic acid having 1-8 carbons, or an ester of a phenol ora naphthol that is further substituted one or more times by nitro,sulfo, carboxy, alkali or alkaline earth metal salt of sulfo or carboxy,cyano, fluoro, chloro, or trifluoromethyl; or R_(x) is selected so as tomake R a carboxylic acid activated by a carbodiimide having 2-14carbons.
 8. A reactive dye, as claimed in claim 7, wherein R_(x) isselected so as to make R an ester of a phenol or a naphthol that isfurther substituted one or more times by nitro, sulfo, carboxy, alkalior alkaline earth metal salt of sulfo or carboxy, cyano, fluoro, chloro,or trifluoromethyl.
 9. A reactive dye, as claimed in claim 7, wherein Rhas the formula —(C═O)—R_(x), wherein R_(x) is chloride, fluoride, orazide; or R_(x) is selected so as to make R a succinimidyl ester,sulfosuccinimidyl ester, or an alkali or alkaline earth metal salt ofsulfosuccinimidyl ester.
 10. A reactive dye, as claimed in claim 7,wherein R_(x) is selected so as to make R a succinimidyl ester,sulfosuccinimidyl ester, or an alkali or alkaline earth metal salt ofsulfosuccinimidyl ester.
 11. A reactive dye, as claimed in claim 10,wherein n=4 or
 5. 12. A reactive dye, as claimed in claim 10, whereinR_(x) is selected so as to make R a succinimidyl ester.
 13. A reactivedye, as claimed in claim 12, having the formula


14. A kit for the fluorescent labeling of an organic substance, wheresaid substance has at least one functional group that is an amine, athiol, an alcohol, a phenol, an aldehyde, a ketone, a phosphate, animidazole, a hydrazine, a hydroxylamine, a disubstituted amine, atrisubstituted amine, a halide, an epoxide, a sulfonate ester, or acarboxylic acid; comprising: a) a reactive dye of the formula

 or the formula

 wherein R2 is H, C1-C6 alkyl or C1-C6 acyl; and n=2 to 8; and R is ahalomethylbenzamidyl, maleimidyl,(3,5-dichloro-2,4,6-triazin-1-yl)amino, isocyanato or an isothiocyanato;or n=1 to 7; and R is a carboxylic acid, an alkali or alkaline earthmetal salt of a carboxylic acid, or R is —(C═O)—R_(x), where R_(x) is achloride, fluoride or azide; or R_(x) is selected so as to make R ahydroxybenzotriazolyl ester, a succinimidyl ester, a sulfosuccinimidylester, an alkali or alkaline earth metal salt of a sulfosuccinimidylester, a symmetric anhydride that links two sulforhodamine 101fluorophores, a mixed anhydride of a sulforhodamine 101 fluorophore anda chloroformate having 2-8 carbons, a mixed anhydride of asulforhodamine 101 fluorophore and a carboxylic acid or perfluorinatedcarboxylic acid having 2-8 carbons, a mixed anhydride of asulforhodamine 101 fluorophore and a sulfonic or fluorinated sulfonicacid having 1-8 carbons, or an ester of a phenol or a naphthol that isfurther substituted one or more times by nitro, sulfo, carboxy, alkalior alkaline earth metal salt of sulfo or carboxy, cyano, fluoro, chloro,or trifluoromethyl; or R_(x) is selected so as to make R a carboxylicacid activated by a carbodiimide having 2-14 carbons; such that R willspontaneously react with said functional group to yield a labeledsubstance; and b) instructions for combining the reactive dye with thesubstance and recovering the labeled substance.
 15. A kit, as claimed inclaim 14, wherein said substance is an amino acid, an amino acidpolymer, a nucleotide, an oligonucleotide, an antisense oligonucleotide,a nucleic acid polymer, or a carbohydrate.
 16. A kit, as claimed inclaim 14, wherein said substance is an amino acid polymer.
 17. A kit, asclaimed in claim 14, wherein said substance is an oligonucleotide ornucleic acid polymer.
 18. A kit, as claimed in claim 14, wherein saidsubstance is a carbohydrate.
 19. A kit, as claimed in claim 14, whereinsaid functional group is an amine or a thiol.